A site-directed mutagenesis interrogation of the carboxy-terminal end of Arabidopsis thaliana threonine dehydratase/deaminase reveals a synergistic interaction between two effector-binding sites and contributes to the development of a novel selectable marker

A site-directed mutagenesis interrogation of the carboxy-terminal end of Arabidopsis thaliana... We fused four mutant omr1 alleles, encoding feedback-insensitive forms of Arabidopsis thaliana biosynthetic threonine dehydratase/deaminase (TD), to the CaMV 35S promoter and transformed these constructs into A. thaliana Columbia wild type plants. The mutant TD forms consisted of our previously isolated double mutant, omr1-1, and three new site-directed mutants, omr1-5, omr1-7, and omr1-8with single point mutations. We employed site-directed mutagenesis to assay the effects of amino acid substitutions in separate regulatory regions within the carboxy-terminal (C-term) allosteric end. TD assays and growth resistance to the isoleucine (Ile) toxic analog -O-methylthreonine (OMT) confirmed the desensitization to feedback inhibition and the viability of these mutant omr1 alleles as selectable markers, respectively. Two of the site-directed mutants, omr1-5 and omr1-7, appeared to influence one of the two separate Ile-binding sites and had a notable 13-fold and 15-fold increase in free Ile, respectively. The omr1-8 appeared to influence the other Ile-binding site and resulted in a 2-fold increase in free Ile. The transgenic omr1-1 double mutant affecting both Ile-binding sites, however, displayed a 106-fold increase in free Ile revealing a profound synergistic interplay between these separate Ile-binding sites. While all of the four omr1 alleles conferred resistance to elevated concentrations of OMT, the progeny of omr1-1 initial transformants exhibited a bushy phenotype at the rosette stage. On the other hand, progeny of transformants omr1-5, omr1-7, and omr1-8 had a normal phenotype, undistinguishable from wild type. Therefore, alleles omr1-5, omr1-7, and omr1-8, proved to be ideal as environmentally-friendly, dominant, selectable markers for plant transformation. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

A site-directed mutagenesis interrogation of the carboxy-terminal end of Arabidopsis thaliana threonine dehydratase/deaminase reveals a synergistic interaction between two effector-binding sites and contributes to the development of a novel selectable marker

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Publisher
Kluwer Academic Publishers
Copyright
Copyright © 2004 by Kluwer Academic Publishers
Subject
Life Sciences; Biochemistry, general; Plant Sciences; Plant Pathology
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1007/s11103-004-0500-z
Publisher site
See Article on Publisher Site

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